U.S. patent number 5,371,624 [Application Number 07/982,468] was granted by the patent office on 1994-12-06 for reflected fluorescence microscope.
This patent grant is currently assigned to Olympus Optical Co., Ltd.. Invention is credited to Masaaki Iwase, Kazuo Kajitani, Takashi Nagano, Keiji Shimizu.
United States Patent |
5,371,624 |
Nagano , et al. |
December 6, 1994 |
Reflected fluorescence microscope
Abstract
A reflected fluorescence microscope is used to observe a
fluorescent image of a stained specimen by reflected illumination,
and includes a light source, an excitation filter for converting
light from the light source into a plurality of narrow-band
excitation lights, a dichroic mirror for reflecting the narrow-band
excitation lights emerging from the excitation filter toward the
specimen and transmitting therethrough the light emerging from the
specimen, an absorption filter for absorbing an extra wavelength
component from light emerging from the dichroic mirror and
transmitting therethrough a wavelength component of the fluorescent
image, an optical system for forming the fluorescent image of the
specimen from the wavelength component transmitted through the
absorption filter, and a transmission wavelength shifting filter
for adjusting the ratio of the quantities of light in units of
fluorochromes.
Inventors: |
Nagano; Takashi (Tokyo,
JP), Shimizu; Keiji (Tokyo, JP), Kajitani;
Kazuo (Tokyo, JP), Iwase; Masaaki (Tokyo,
JP) |
Assignee: |
Olympus Optical Co., Ltd.
(Tokyo, JP)
|
Family
ID: |
26336572 |
Appl.
No.: |
07/982,468 |
Filed: |
November 27, 1992 |
Foreign Application Priority Data
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Nov 29, 1991 [JP] |
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3-317100 |
Jan 10, 1992 [JP] |
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4-003076 |
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Current U.S.
Class: |
359/389; 359/368;
359/385 |
Current CPC
Class: |
G02B
21/082 (20130101); G02B 21/16 (20130101) |
Current International
Class: |
G02B
21/08 (20060101); G02B 21/06 (20060101); G02B
021/06 () |
Field of
Search: |
;359/368-390,885,887-891 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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|
164680 |
|
Dec 1985 |
|
EP |
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2188447 |
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Sep 1987 |
|
EP |
|
1447166 |
|
Aug 1976 |
|
GB |
|
Primary Examiner: Sugarman; Scott J.
Assistant Examiner: Nguyen; Thong
Attorney, Agent or Firm: Frishauf, Holtz, Goodman &
Woodward
Claims
What is claimed is:
1. A reflected fluorescence microscope having an optical system,
for observing a fluorescent image of a stained specimen illuminated
by reflected illumination, comprising:
light source means for supplying a light to said optical system for
the reflected illumination;
excitation light generating means, disposed on an optical path
between said light source means and said optical system, for
converting the light from said light source means into a plurality
of narrow-band excitation lights each having narrow bands
components;
a dichroic mirror, disposed on an optical path of said optical
system on which the narrow-band excitation lights from said
excitation light generating means and the fluorescent image from
said specimen are incident, said dichroic mirror reflecting to the
specimen the narrow-band excitation lights emerging from said
excitation light generating means and transmitting therethrough the
fluorescent image from the specimen that emits a plurality of types
of fluorescences upon being excited by the narrow-band excitation
lights;
absorption filter means for absorbing an extra wavelength component
from the fluorescent image emerging from said dichroic mirror;
and
filter means, disposed on said optical path between said optical
system and said light source means, for varying a ratio in
intensity between a pair of narrow-band excitation lights included
in the plurality of narrow-band excitation lights, thereby
effecting switching between at least a first state and a second
state, a wavelength of one of said pair of narrow-band excitation
lights being shorter than a wavelength of the other of said pair of
narrow-band excitation lights, and wherein said filter means in the
first state has first transmittance characteristics having a low
transmittance at a wavelength band of said one of said pair of
narrow-band excitation lights and a high transmittance at a
wavelength band of said other of said pair of narrow-band
excitation lights, and said filter means in the second state has
second transmittance characteristics having a high transmittance at
a wavelength band of said one of said pair of narrow-band
excitation lights and a low transmittance at a wavelength band of
said other of said pair of narrow-band excitation lights.
2. A microscope according to claim 1, wherein said filter means is
selectively changeable between first transmittance characteristics
wherein the transmittance is continuously increased from a short
wavelength side to a long wavelength side within a predetermined
wavelength range to form a first transmission wavelength band, and
second transmittance characteristics wherein the transmittance is
continuously decreased from the long wavelength side to the short
wavelength side within a predetermined wavelength range to form a
second transmission wavelength band.
3. A microscope according to claim 2, wherein said filter means
includes:
a plurality of filters each having different transmittance
characteristics, said plurality of filters including at least a
first filter having the first transmittance characteristics and a
second filter having the second transmittance characteristics;
and
a selecting mechanism for selectively changing said plurality of
filters.
4. A microscope according to claim 3, wherein said selecting
mechanism includes:
a tube body constituting part of a light projection tube that forms
the optical path between said light source means and said dichroic
mirror;
a slider member held by said tube body to be slidable in a
direction perpendicular to an optical axis;
a plurality of mounting portions linearly formed in said slider
member to extend in a sliding direction of said slider member and
on which said filters are mounted; and
a positioning mechanism for positioning said slider member at a
position where each of said filters coincides with the optical
axis.
5. A microscope according to claim 1, wherein said filter means
includes an interference filter, the transmission wavelength band
of which is shifted in accordance with an incident angle of
narrow-band light, and a rotary mechanism for rotatably supporting
said interference filter so as to change the incident angle of the
narrow-band light.
6. A microscope according to claim 5, wherein said rotary mechanism
includes a tube body constituting part of a light projection tube
that forms the optical path between said light source means and
said dichroic mirror, a filter frame, fitted in said tube body, for
rotatably holding said interference filter in an axis perpendicular
to an optical axis, a pin member movable to push said filter frame,
a spring member for pushing said pin member, and a spring
receptacle member, fixed to said tube body, for supporting said pin
member and said spring member.
7. A microscope according to claim 1, wherein said filter means
comprises an interference filter having transmittance
characteristics and forming a trapezoidal transmission wavelength
band, and wherein the trapezoidal transmission wavelength band is
shifted by inclining said interference filter.
8. A reflected fluorescence microscope, having an optical system,
for observing a fluorescent image of a stained specimen illuminated
by reflected illumination, comprising:
light source means for supplying a light to said optical system
used for the reflected illumination;
at least one fluorescent filter unit including an excitation
filter, disposed on an optical path between said light source means
and said optical system, for converting the light from said light
source means into a plurality of narrow-band excitation lights, a
dichroic mirror disposed on an optical path of said optical system
on which the narrow-band excitation lights from said excitation
filter and the fluorescent image lights; from said specimen are
incident, said dichroic mirror reflecting the narrow-band
excitation lights emerging from said excitation filter to the
specimen and transmitting therethrough the fluorescent image from
the specimen that emits a fluorescence upon being excited by the
narrow-band excitation lights, and an absorption filter disposed on
said optical path of said optical system on which the fluorescent
image emerging from said dichroic mirror are incident, said
dichroic mirror absorbing an extra wavelength component from the
fluorescent image and transmitting therethrough a wavelength
component of the fluorescent image, said excitation and absorption
filters each comprising interference filters each formed of a
dielectric multilayer film and each being supported to be pivotal
in an axis perpendicular to an optical axis; and
an interlock mechanism for pivoting said excitation and absorption
filters of said at least one fluorescent filter unit in an
interlocked manner.
9. A microscope according to claim 8, further comprising a
selecting mechanism, having a plurality of said fluorescent filter
units, for selectively inserting and removing respective ones of
said fluorescent filter units in and from an optical path.
10. A microscope according to claim 9, wherein said selecting
mechanism comprises a turret mechanism having a rotating axis
parallel to the optical path and radially holding said plurality of
fluorescent filter units within a plane perpendicular to the
rotating axis.
11. A microscope according to claim 9, wherein said selecting
mechanism comprises a slider mechanism, held to be slidable in a
direction perpendicular to the optical path, for holding said
plurality of fluorescent filter units in a sliding direction
thereof.
12. A microscope according to claim 8, wherein said interlock
mechanism comprises:
a first gear having a rotation center to which a rotating shaft of
said excitation filter is fixed;
a second gear having a rotation center to which a rotating shaft of
said absorption filter is fixed; and
an intermediate gear meshing with both of said first and second
gears.
13. A microscope according to claim 8, wherein said interlock
mechanism comprises:
a first pulley having a rotation center to which a rotating shaft
of said excitation filter is fixed;
a second pulley having a rotation center to which a rotating shaft
of said absorption filter is fixed; and
a coupling belt looped between said first and second pulleys.
14. A microscope according to claim 8, wherein said interlock
mechanism comprises driving means for driving rotating shafts of
said excitation and absorption filters in electrical synchronism
with each other.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a reflected fluorescence
microscope which is utilized to observe a living tissue or cell in
the fields of medicine, biology, and the like, and which has an
excitation filter and an absorption filter and, more particularly,
to a reflected fluorescence microscope in which the excitation
wavelength and the absorption wavelength can be adjusted.
2. Description of the Related Art
Generally, reflected fluorescence microscopes are widely used in
medicine, biology, and other fields to detect a protein, a gene,
and the like marked with a fluorescent label on a living tissue or
cell.
In recent years, as various types of fluorochromes have been
developed, the reflected fluorescence microscopes are used
particularly to study the mutual positional relationship among
specific substances and the localization of a specific substance in
a cell by means of multiple stain using various types of materials
as the fluorescent labels.
When specimens stained with various fluorescent materials are to be
observed using a reflected fluorescence microscope of this type, a
fluorescent filter set consisting of an excitation filter, a
dichroic mirror, and an absorption filter is prepared for each
specific fluorochrome, and an optimum combination of the excitation
and absorption wavelength regions is obtained by selectively using
the fluorescent filter sets, thereby observing the specific
fluorochrome.
In this manner, the conventional reflected fluorescence microscope
has fixed excitation and absorption wavelength regions for each
fluorescent filter set. Therefore, in order to observe various
types of specimens stained with fluorochromes with the optimum
excitation and absorption wavelength regions, a considerably large
number of fluorescent filter sets are needed. Then, the number of
components is increased, leading to an increase in manufacturing
costs.
When the mutual positional relationship among the multiple
fluorochromes that stain the fluorescent specimen is to be studied,
the respective fluorescent images of the fluorescent specimen must
be recorded on a photograph or a video memory in multiple exposure.
Therefore, the conventional reflected fluorescence microscope is
not suitable for detection of fluorochromes having high
discoloration speeds or detection of the mutual positional
relationship among the fluorochromes by using time as a
parameter.
When the fluorescent filter sets are selectively used,
off-centering of the observation optical system occurs depending on
the component precision of the dichroic mirror, the absorption
filter, and the like, and an error occurs in the mutual positional
relationship detected from the fluorescent image.
SUMMARY OF THE INVENTION
The present invention has been made to solve the problems described
above, and has as its object to provide a reflected fluorescence
microscope which can adjust the spectral transmittance
characteristics of fluorescent filter sets to optimum values
according to the fluorochromes of the specimen without exchanging
excitation and absorption filters.
It is another object of the present invention to provide a
reflected fluorescence microscope capable of observing a
fluorescent specimen stained with multiple fluorochromes without
exchanging a fluorescent filter set and capable of obtaining a
fluorescent image that can be observed easily by appropriately
changing the fluorescent intensities of the respective
fluorochromes with a simple operation.
According to the present invention, there is provided a reflected
fluorescence microscope for observing a fluorescent image of a
stain specimen by reflected illumination, comprising:
a light source for supplying light used for the reflected
illumination;
an excitation filter for converting the light from the light source
into a plurality of narrow-band excitation lights each having
narrow bands;
a dichroic mirror, disposed at a position where the lights from the
excitation filter and a light from the specimen are incident
thereon, for reflecting the narrow-band excitation lights emerging
from the excitation filter toward the specimen and for transmitting
therethrough the light emerging from the specimen that emits a
plurality of types of fluorescences upon being excited by the
narrow-band excitation lights;
an absorption filter for absorbing or reflecting an extra
wavelength component from light emerging from the dichroic mirror
and transmitting therethrough a wavelength component of the
fluorescent image;
an optical system for forming the fluorescent image of the specimen
from the wavelength component transmitted through the absorption
filter; and
an interference filter, disposed on an optical path through which
the light supplied from the light source passes until being
incident on the dichroic mirror, and having a variable transmission
wavelength band defined by a transmittance and a wavelength, for
selectively causing, with respect to a pair of narrow-band
excitation lights among the plurality of narrow-band excitation
lights, a first state, wherein the interference filter has partly
low transmittance characteristics on a short wavelength side of one
narrow-band excitation light and high transmittance characteristics
on a long wavelength side of the other narrow-band excitation
light, and a second state, wherein the interference filter has
partly low transmittance characteristics on a long wavelength side
of the other narrow-band excitation light and high transmittance
characteristics on a short wavelength side of one narrow-band
excitation light.
More specifically, the transmission wavelength shifting filter has
transmittance characteristics for forming a trapezoidal
transmission wavelength band, and the transmission characteristics
are changed so that the transmission wavelength band is shifted in
a direction of the wavelength while a shape thereof is essentially
maintained.
According to the present invention having the arrangement as
described above, the interference filter is held in the reflected
illumination optical path before the dichroic mirror to be
rotatable in an axis perpendicular to the optical axis. When this
filter is inclined with respect to the optical axis, the
transmittance characteristics of the interference filter are
changed so that, in the transmission wavelength band, part of the
long-wavelength side narrow band is gradually cut and that the
interference filter has a high transmittance in the low-wavelength
side band.
As a result, the quantities of light of the plurality of
narrow-band excitation lights emerging from the excitation filter
are changed, and accordingly the ratio of the intensity of the
plurality of fluorescences emitted from the specimen is changed.
Therefore, the brightnesses of a plurality of types of fluorescent
images can be easily adjusted, and efficient excitation can be
performed.
Alternatively, a transmission wavelength shifting filter having
first transmittance characteristics wherein the transmittance is
continuously increased from the short wavelength side to the long
wavelength side within a predetermined wavelength range to form a
first transmission wavelength band, and a second transmission
wavelength shifting filter having second transmittance
characteristics wherein the transmittance is continuously decreased
from the long wavelength side to the short wavelength side within a
predetermined wavelength range to form a second transmission
wavelength band, are selectively changed.
According to the present invention having the arrangement as
described above, the transmission wavelength bands can be changed
by selectively changing a first filter having a high transmittance
with respect to short wavelength-side narrow-band excitation light
and a low transmittance with respect to long wavelength-side
narrow-band excitation light, and a second filter having a low
transmittance with respect to short wavelength-side narrow-band
excitation light and a high transmittance with respect to long
wavelength-side narrow-band excitation light.
Furthermore, according to the present invention, there is also
provided a reflected fluorescence microscope, comprising:
a light source for supplying light used for reflected
illumination;
at least one fluorescent filter unit for changing transmittance
characteristics in accordance with a wavelength component of the
fluorescent image,
the fluorescent filter unit having an excitation filter for
converting the light from the light source into a narrow-band
excitation light, a dichroic mirror, disposed at a position where
the light from the excitation filter and the Light from the
specimen are incident thereon, for reflecting the narrow-band
excitation light emerging from the excitation filter toward the
specimen and for transmitting therethrough the light emerging from
the specimen that emits a fluorescence upon being excited by the
narrow-band excitation light, and an absorption filter for
absorbing an extra wavelength component from light emerging from
the dichroic mirror and transmitting therethrough a wavelength
component of the fluorescent image, and
the excitation and absorption filters each being constituted by an
interference filter formed of a dielectric multilayer film and each
being supported to be pivotal about an axis perpendicular to an
optical axis;
an interlock mechanism for pivoting the excitation and absorption
filters of the fluorescent filter unit in an interlocked manner;
and
an optical system for forming the fluorescent image of the specimen
from a wavelength component transmitted through the absorption
filter.
According to the present invention having the arrangement as
described above, the excitation and absorption filters are
interlocked through the interlock mechanism and can respectively be
inclined at predetermined amounts with respect to the corresponding
optical axes. When the excitation and absorption filters are
inclined from, e.g., a state perpendicular to the optical axes, the
incident angles of light incident on these filters are gradually
increased. When the incident angles are increased, the transmission
wavelength band is shifted.
Therefore, when the maximum transmittance wavelengths of the
excitation and absorption filters are set at appropriate values and
the excitation and absorption filters are pivoted in accordance
with the fluorochromes of the specimen, optimum excitation and
absorption wavelength ranges can be set without exchanging the
fluorescent filter set.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a view showing the arrangement of a reflected
fluorescence microscope according to the first embodiment of the
present invention;
FIG. 2 is a sectional view showing the rotary holding mechanism of
an interference filter provided to the reflected fluorescence
microscope of the first embodiment;
FIG. 3 is a graph showing the transmission wavelength band of an
excitation filter obtained when an interference filter is not
provided;
FIG. 4A is a graph showing the transmittance characteristics
obtained when the interference filter is inclined to the long
wavelength side and the wavelength of the excitation light obtained
when both interference and excitation filters are used;
FIG. 4B is a graph showing the transmittance characteristics
obtained when the interference filter is inclined to the short
wavelength side and the wavelength of the excitation light obtained
when both interference and excitation filters are used;
FIG. 5A is a view showing a filter selecting mechanism used in a
reflected Fluorescence microscope according to the second
embodiment of the present invention, and explaining positioning of
the filter;
FIG. 5B is a sectional view of the filter selecting mechanism used
in the reflected fluorescence microscope according to the second
embodiment of the present invention;
FIG. 6A is a graph showing the filter characteristics of a filter
having a high transmittance in the short wavelength side and a low
transmittance in the long wavelength side, and the wavelength of
excitation light when such a filter and an excitation filter are
used;
FIG. 6B is a graph showing the filter characteristics of a filter
having a low transmittance in the short wavelength side and a high
transmittance in the long wavelength side, and the wavelength of
excitation light when such a filter and an excitation filter are
used;
FIG. 7 is a graph showing the transmittance characteristics of a
fluorescent filter set;
FIG. 8 is a view showing the arrangement of the optical system of a
reflected fluorescence microscope according to the third embodiment
of the present invention;
FIG. 9 is a sectional view of a fluorescent filter set provided to
the reflected fluorescence microscope according to the third
embodiment;
FIG. 10 is a graph showing the transmittance characteristics of the
fluorescent filter set shown in FIG. 9;
FIG. 11 is a graph showing a shift in transmittance characteristics
caused by moving the fluorescent filter set shown in FIG. 9;
FIG. 12 is a view showing the first modification of a portion
associated with the fluorescent filter set;
FIG. 13 is a sectional view of the first modification;
FIG. 14 is a view showing the second modification of the portion
associated with the fluorescent fluorescence set;
FIG. 15 is a sectional view of the second modification; and
FIG. 16 is a view showing the third modification of the portion
associated with the fluorescent filter set.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The preferred embodiments of the present invention will be
described below with reference to the accompanying drawings.
FIG. 1 is a view showing the arrangement of the optical system of a
reflected fluorescence microscope according to the first embodiment
of the present invention. Reference numeral 11 denotes a light
source, e.g., a mercury lamp; 12, a collector lens for collecting
light projected by the light source 11; and 13, an interference
filter serving as a rotatably held transmission wavelength shifting
filter which has a rotation axis perpendicular to the output
optical axis of the collector lens 12. An aperture stop 14, a field
stop 15, an excitation filter 16 having transmittance
characteristics shown in FIG. 7, and a dichroic mirror 17 having
transmittance characteristics shown in FIG. 7 are disposed on the
output optical axis of the interference filter 13 in this
order.
Reference numeral 18 denotes an objective lens; and 19, a
vertically movable stage on which a specimen 20 is placed. Incident
illumination light emerging from the dichroic mirror 17 is radiated
on the specimen 20 through the objective lens 18.
Object light (fluorescent image) emerging from the specimen 20 is
guided to the dichroic mirror 17 through the objective lens 18
again. An absorption filter 21 having transmittance characteristics
shown in FIG. 7 and a beam splitter 22 are disposed on the exit
side of the dichroic mirror 17. The beam splitter 22 is removably
inserted in the optical path to switch the optical path between the
observation and photographing systems as required. An eyepiece
optical system 23 is disposed on the observation optical path of
the beam splitter 22, and a photographing eyepiece 24 is disposed
on the photographing optical path of the beam splitter 22.
The excitation filter 16, the dichroic mirror 17, and the
absorption filter 21 constitute a fluorescent filter set. The
excitation filter 16 has two high-transmittance regions
.lambda..sub.EA and .lambda..sub.EB for effectively exciting two
types of fluorochromes A and B, as shown in FIG. 7. Each of the
dichroic mirror 17 and the absorption filter 21 has a
high-transmittance region falling between the two regions
.lambda..sub.EA and .lambda..sub.EB and a high-transmittance region
of a transmittance higher than that of the region .lambda..sub.EB.
The specimen 20 is stained with multiple fluorochromes to emit two
types of fluorescences upon excitation by excitation lights having
central wavelengths at wavelengths .lambda..sub.EA and
.lambda..sub.EB.
FIG. 2 is a sectional view showing the rotary holding mechanism of
the interference filter 13 adopted in the reflected fluorescence
microscope according to the present invention. The rotary holding
mechanism is provided to a portion of a reflected illumination
projection tube that transmits the illumination light of the light
source 11 to the fluorescent filter set.
More specifically, the rotary holding mechanism is constituted by a
tube body 31 forming part of the projection tube, a filter frame 32
fitted in the tube body 31 to rotatably hold the interference
filter 13 in an axis perpendicular to the optical axis, a pin 33
capable of pushing the filter frame 32, a coil spring 34 for
pushing the pin 33, a spring receptacle 35, fixed to the tube body
31, for supporting the pin 33 and the coil spring 34, and the
like.
In the rotary holding mechanism, the interference filter 13 is
rotated by an external electrical or mechanical, and the pin 33
applies an urging force to fix the filter frame 32, i.e., the
interference filter 13 at an arbitrary angular position upon
reception of the urging force from the coil spring 34.
The operation of the reflected fluorescence microscope having the
structure as described above will be described.
When the interference filter 13 does not exist, light projected by
the light source 11 is collected by the collector lens 12, and is
incident on the excitation filter 16. Only a plurality of lights
each having narrow-band excitation light wavelengths, as shown in
FIG. 3, are transmitted through the excitation filter 16, are
reflected by the dichroic mirror 17, and are radiated on the
specimen 20 on the stage 19. Since the specimen 20 is stained with
two types of fluorochromes in advance, it emits two types of
fluorescences upon excitation by the light having two excitation
wavelength bands .lambda..sub.EA and .lambda..sub.EB, as shown in
FIG. 3.
The fluorescences emitted from the specimen 20 are transmitted
through the objective lens 18 and the dichroic mirror 17. Light
components having unnecessary wavelength bands are absorbed or
reflected by the absorption filter 21, and only a wavelength of the
fluorescent image is extracted. This fluorescent image is guided to
the observation eyepiece optical system or photographing
system.
Due to a difference in fluorescent intensity among the respective
fluorochromes, it is difficult to balance the two types of
fluorescent intensities to obtain a desired brightness.
Conventionally, in order to adjust the fluorescent intensities of
the two types of the fluorochromes, for example, the
characteristics of the absorption filter 21 in the observation
optical path are varied, or an auxiliary filter is inserted in the
observation optical path to partially cut the fluorescence emitted
by the specimen 20. Alternatively, an ND filter having a neutral
density and not exhibiting selective spectral absorption is
inserted in the illumination optical path, or the aperture stop 14
is adjusted.
With the former method of cutting fluorescence, however, the
excitation light for exciting the fluorescence which is eventually
cut becomes wasteful. Then, the fluorescence utilization efficiency
is degraded by this wasteful light, and the specimen 20 is
subjected to an extra damage, which is not preferable. On the other
hand, with the latter method of adjusting the light intensity,
since the intensities of the excitation light components having two
wavelengths cannot be adjusted independently of each other, the
ratio of the intensity of the two types of fluorescences cannot be
changed.
Hence, according to the present invention, the interference filter
13 for shifting the transmission wavelength which has the rotary
holding mechanism as shown in FIG. 2 is inserted in the reflected
illumination light path, thereby obtaining illumination light
components having wavelengths as shown in FIGS. 4A and 4B. More
specifically, the transmission wavelength shifting filter 13 is an
interference filter, and the transmittance characteristics as shown
in FIG. 4A can be obtained when the interference filter 13 is
inserted perpendicularly (an inclination of 0.degree.) with respect
to the output optical axis of the collector lens 12. On the other
hand, when the interference filter 13 inserted to be inclined at
45.degree. with respect to an axis perpendicular to the output
optical axis of the collector lens 12, the transmittance
characteristics as shown in FIG. 4B can be obtained.
Generally, the conditions for interference of an interference
filter can be represented by the following equation:
where .lambda. is the maximum transmittance wavelength, t is the
optical thickness (including the phase difference caused in the
interface of the dielectric by conversion into an optical path
length) of the dielectric, and .PHI. is the reflection angle of the
interface.
When the degree m is constant and the conditions for interference
are constant, the wavelength .lambda. is proportional to cos.PHI..
.PHI. is a reflection angle and can be considered equivalent to the
incident angle since it is conjugate to the incident angle from
Snell's law.
When the incident angle is increased, cos.PHI. is decreased, the
wavelength .lambda. is also decreased simultaneously, and thus the
maximum transmittance is gradually shifted to the low wavelength
side. Therefore, when the interference filter 13 is gradually
inclined from the axis perpendicular to the optical axis up to
45.degree., the transmittance wavelength band can be continuously
shifted from the band as shown in FIG. 4A to the band as shown in
FIG. 4B.
Furthermore, when both the interference filter 13 which is set in a
direction (an inclination of 0.degree.) perpendicular to the
optical axis and the excitation filter 16 are employed, the
obtained excitation light has the wavelengths indicated by the
hatched portions in FIG. 4A. As is apparent from FIG. 4A, in the
short excitation wavelength band .lambda..sub.EA, part of the
excitation light obtained by the excitation filter 16 which is in
the short wavelength side is cut. On the other hand, in the long
excitation wavelength band .lambda..sub.EB, the ratio of the
intensity of the two excitation wavelength bands .lambda..sub.EA
and .lambda..sub.EB is changed since there is no influence of the
interference filter 13.
As a result, the ratio of the intensity of the two types of
fluorescences excited by the two excitation wavelength bands
.lambda..sub.EA and .lambda..sub.EB can be changed.
When both the interference filter 13 which is set to be inclined at
45.degree. from the axis perpendicular to the optical axis and the
excitation filter 16 are employed, the obtained excitation light
has the wavelengths indicated by the hatched portions in FIG. 4B.
In this case, in the long excitation wavelength band
.lambda..sub.EB, of the excitation light obtained by the excitation
filter 16, part of the excitation light obtained by the excitation
filter 16 which is in the long wavelength side is cut. On the other
hand, in the short excitation wavelength band .lambda..sub.EA, the
ratio of the intensity of the two excitation wavelength bands
.lambda..sub.EA and .lambda..sub.EB can be changed since there is
no influence of the interference filter 13. As a result, the ratio
of the intensity of the two types of fluorescences excited by the
two excitation wavelength bands .lambda..sub.EA and .lambda..sub.EB
can be changed.
When the inclination of the interference filter 13 is gradually
changed between the state of 0.degree. and the state of 45.degree.,
that is, when the interference filter 13 is gradually inclined from
0.degree., as the state shown in FIG. 4A, toward 45.degree., the
transmittance band of the interference filter 13 is shifted to the
short-wavelength side in accordance with the angle of inclination.
At this time, in the short wavelength band .lambda..sub.EA, the
region which is cut is gradually decreased, and the intensity of
excitation light is increased. In the long excitation wavelength
band .lambda..sub.EB, since the wavelength of the excitation light
partially overlaps the non-transmitting range of the interference
filter 13, the region which is cut is gradually increased from the
long wavelength side, and accordingly the intensity of excitation
light is decreased.
Hence, as is apparent from the above description, the ratio of the
intensities of the excitation lights having the bands
.lambda..sub.EA and .lambda..sub.EB can be continuously changed.
Accordingly, the ratio of the intensity of the two types of
fluorescences excited by the excitation wavelength bands
.lambda..sub.EA and .lambda..sub.EB can be continuously changed.
Thus, when the inclination of the interference filter 13 is set at
an arbitrary angle between the two states of 0.degree. and
45.degree., the ratio of the intensities of the two types of
fluorescences excited by the excitation wavelength bands
.lambda..sub.EA and .lambda..sub.EB can be adjusted to a desired
value.
Assume that a difference occurs in fluorescent intensity by two
types of fluorochromes, or that a difference occurs in
discoloration speed between two types of fluorescences. In this
case, the balances in brightness of the two types of fluorescent
images are different. Then, with the arrangement of the embodiment
as described above, the ratio of the fluorescent intensities of the
two types of fluorescences excited by excitation wavelength bands
.lambda..sub.EA and .lambda..sub.EB can be adjusted to a desired
value by changing the inclination of the interference filter 13,
i.e., by a simple operation, so that a problem such as one
fluorescent image is excessively bright or dark in observation or
photographing can be solved. In addition, since this adjustment can
be performed by controlling illumination light, a degradation in
excitation efficiency as described above will not occur, and no
extra damage will be applied to the specimen 20.
FIGS. 5A and 5B are views for explaining the second embodiment of
the present invention.
In the second embodiment, a filter selecting mechanism is used in
place of a filter holding mechanism shown in FIG. 2. Referring to
FIG. 5A, reference numeral 41 denotes a tube body constituting part
of the light projection tube; 42, a slider for inserting and
removing a through hole and a plurality of filters in and from the
optical path; 43a, a through hole; 43b and 43c, filters (to be
described later) used in this embodiment; 44a to 44c, positioning
click grooves for the through hole 43a and the filters 43a and 43b,
respectively; 45, a pin for fitting and fixing the slider 42 by
fitting it in the click holes 44a to 44c; 46, a coil spring for
pressing the pin 45; and 47, a spring receptacle, fixed to the tube
body 41, for supporting the pin 45 and the coil spring 46.
When the filters 43b and 43c having the filter mechanisms as
described above are combined with the excitation filter 16, the
obtained excitation light appears to have wavelengths as indicated
by the hatched portions in FIGS. 6A and 6B.
Especially, in FIG. 6A, since the filter 43b has a high
transmittance with respect to the short excitation wavelength band
.lambda..sub.EA and a low transmittance with respect to the long
excitation wavelength band .lambda..sub.EB, which two types of
excitation wavelength bands .lambda..sub.EA and .lambda..sub.EB
being obtained by the excitation filter 16, of the intensities of
the two types of excitation wavelength bands .lambda..sub.EA and
.lambda..sub.EB, the intensity of the long excitation wavelength
band .lambda..sub.EB is lower than that of the short excitation
wavelength band .lambda..sub.EA. As a result, the ratio of the
fluorescent intensities of the two types of fluorescences excited
by the excitation wavelength bands .lambda..sub.EA and
.lambda..sub.EB changes at a predetermined rate in accordance with
a change in ratio of the excitation light intensities when compared
to the ratio of the same obtained when the filter 43b is not
inserted in the optical path.
In FIG. 6B, since the filter 43c has a low transmittance with
respect to the short excitation wavelength band .lambda..sub.EA and
a high transmittance with respect to the long excitation wavelength
band .lambda..sub.EB, which two types of excitation wavelength
bands .lambda..sub.EA and .lambda..sub.EB being obtained by the
excitation filter 16, of the intensities of the two types of
excitation wavelength bands .lambda..sub.EA and .lambda..sub.EB,
the intensity of the long excitation wavelength band
.lambda..sub.EB is higher than that of the short excitation
wavelength band .lambda..sub.EA. As a result, the ratio of the
fluorescent intensities of the two types of fluorescences excited
by the excitation wavelength bands .lambda..sub.EA and
.lambda..sub.EB changes at a predetermined rate in accordance with
a change in ratio of the excitation light intensities when compared
to the ratio of the same obtained when the filter 43c is not
inserted in the optical path.
Therefore, when the through hole 43a and the filters 43b and 43c
are appropriately selected by the slider 42, the ratio of the
intensities of the excitation lights having the bands
.lambda..sub.EA and .lambda..sub.EB can be appropriately changed at
a predetermined rate from the value obtained when only the
excitation filter 16 is used to the ratio represented by the
hatched portions in FIG. 6A or to the ratio represented by the
hatched portions in FIG. 6B, and the ratio of the intensities of
the two types of fluorescences excited by the excitation wavelength
bands .lambda..sub.EA and .lambda..sub.EB, which ratio being
equivalent to the ratio of the intensities of the excitation lights
having the bands .lambda..sub.EA and .lambda..sub.EB, can be
adjusted at an arbitrary predetermined rate.
Since the gradient of the transmittance characteristics of the
filters 43b and 43c can be arbitrarily designed, the ratio of the
intensities of the two types of fluorescences excited by the
excitation wavelength bands .lambda..sub.EA and .lambda..sub.EB can
be freely set by the design of the employed filters 43b and 43c in
accordance with the fluorochromes and the state of the specimen
20.
Therefore, according this embodiment having this arrangement, when
the difference in fluorescent intensity or discoloration state
between two types of fluorochromes is known, the ratio of the
intensities of the two types of fluorescences can be quickly
changed only by switching the slider 42, and a fluorescent image
which is easy to observe can be obtained by eliminating a variation
in brightness of the fluorescent image among the fluorochromes
during observation and photographing. Furthermore, since adjustment
can be performed by controlling illumination light, the degradation
in excitation efficiency as described above will not occur, and no
extra damage will be applied to the specimen 20.
In this embodiment, the two types of filters 43b and 43c are used
in the slider 42. However, a plurality of demands according to the
favor of the user or the state of microscopic observation can be
dealt with by using a plurality of (e.g., one or more) types of a
plurality of filters and sliders corresponding to them.
The third embodiment of the present invention will now be
described.
FIG. 8 shows the arrangement of the optical system of a reflected
fluorescence microscope according to the third embodiment, and FIG.
9 shows a fluorescent filter set provided to this optical system.
The same constituent elements as those in the first and second
embodiments described above are denoted by the same reference
numerals.
In the first and second embodiments, the transmission wavelength
range is shifted by a filter provided midway along the light
projection tube. In the third embodiment, the transmission
wavelength range is shifted by operating an excitation filter and
an absorption filter.
In the reflected fluorescence microscope of the third embodiment,
the beam emitted from a light source 11 is collected by a collector
lens 12 and is incident on an excitation filter 16, having the
transmittance characteristics indicated by a solid line E in FIG.
10, through an aperture stop 14, a field stop 15, and another
collector lens. The beam transmitted through the excitation filter
16 becomes excitation light having a predetermined excitation
wavelength, is incident on a dichroic mirror 17 disposed on the
observation optical axis and having the transmittance
characteristics indicated by a broken line DM in FIG. 10, and is
reflected toward a specimen 20. The excitation light incident on
the observation optical axis is projected onto the specimen 20
through an objective lens 18.
The specimen 20 irradiated with the excitation light emits a
fluorescence having a wavelength longer than that of the excitation
light. The fluorescence from the specimen 20 which has such
wavelength characteristics is incident on the objective lens 18 and
then on an absorption filter 21, having the transmittance
characteristics indicated by an alternate long and short dashed
line B in FIG. 10, through the dichroic mirror 17.
When the transmittance characteristics of the absorption filter 21
are set in accordance with the wavelength characteristics of the
fluorescence emitted by the specimen 20, only the fluorescence is
transmitted through the absorption filter 21. The excitation filter
16, the dichroic mirror 17, and the absorption filter 21 constitute
a fluorescent Filter set 51.
The fluorescence from the specimen 20 passing through the
absorption filter 21 is incident on a prism 22 disposed on the
observation optical axis, and its optical path is branched into an
observation optical system in which an eyepiece 23 is disposed and
a photographing optical system in which a photographing lens 24 is
disposed.
FIG. 9 shows the practical arrangement of the fluorescent filter
set 51. The fluorescent filter set 51 has a columnar through port
at its central portion and a fluorescent cube 60 having an opening
communicating with this through port on its one side surface, as
shown in FIG. 9. The excitation filter 16 is disposed in the
opening of this fluorescent cube 60, the absorption filter 21 is
disposed on one end of the through port, and the dichroic mirror 17
is disposed in a position of the through port opposing the opening
at an angle of 45.degree.. A mounting portion 61 for detachably
mounting the fluorescent filter set 51 to the microscope body is
formed on the other side surface of the fluorescent cube 60.
The excitation and absorption filters 16 and 21 are constituted by
interference filters formed of dielectric multilayer films. The
excitation filter 16 is supported by a rotating shaft 62
perpendicular to the optical axis, and the rotating shaft 62 is
supported on the fluorescent cube 60 by bearings. Thus, the
excitation filter 16 is pivotal about the axis perpendicular to the
optical axis. A gear 63 pivotally supported on the fluorescent cube
60 is mounted to one end of the rotating shaft 62.
The absorption filter 21 is supported on a rotating shaft 64
perpendicular to the optical axis and supported on the fluorescent
cube 60 by bearings. Thus, the absorption filter 21 is also pivotal
about the axis perpendicular to the optical axis. A gear 65
pivotally supported on the fluorescent cube 60 by bearings is
mounted on one end of the rotating shaft 64.
The gear 63 of the excitation filter 16 and the gear 65 of the
absorption filter 21 are coupled to each other through a gear 66
rotatably supported on the fluorescent cube 60 by bearings, thereby
constituting an interlock mechanism.
The angles (incident angles of the beams) of the excitation and
absorption filters 16 and 21 defined with the corresponding optical
axes are changed in the interlocked manner. Note that the gear
speed reducing ratio of the gears 63, 65, and 66 is set such that
the shift amounts of the cutoff wavelengths obtained by inclining
the excitation and absorption filters 16 and 21 with respect to the
corresponding optical axes become equal to each other.
The relationship between the changes in angle of reflected light on
the excitation and absorption filters 16 and 21 and the maximum
transmittance characteristic range will be described. As described
above, when the incident angle of the interference filter is
increased, the maximum transmittance characteristic range is
gradually shifted to the short wavelength side.
For this reason, when the planes of incidence of the excitation and
absorption filters 16 and 21 constituted by the interference
filters having the characteristics as described above are gradually
inclined from the state perpendicular to the corresponding optical
axes, the transmittance characteristics E and B of the excitation
and absorption filters 16 and 21, respectively, are shifted to the
short wavelength side by .DELTA..lambda. to become transmittance
characteristics E' and B', respectively, as shown in FIG. 11.
In the embodiment having the arrangement as described above, when
either one of the gears 63, 65, and 66 is rotated, the remaining
two gears are rotated through the gear 66 in the interlocked
manner, and the excitation and absorption filters 16 and 21
integral with the gears 63 and 65, respectively, are pivoted about
the rotating shafts 62 and 64, respectively.
As a result, the incident angles of the beams incident on the
excitation and absorption filters 16 and 21 are changed, so that
the transmittance characteristics E and B of the excitation and
absorption filters 16 and 21, respectively, are shifted, as
described above.
Accordingly, when a manufacturing error is present in any one of
the excitation and absorption filters 16 and 21 and the dichroic
mirror 17 constituting the fluorescent filter set, or when the
optimum excitation state is changed due to a change in the state of
the specimen 20, the excitation and absorption filters 16 and 21
may be rotated to adjust the corresponding transmittance
characteristics E and B to optimum states, as described above, so
that the fluorescent image of the specimen 20 can always be
observed in the optimum excitation state without exchanging the
fluorescent filter set. Therefore, a degradation in fluorescent
image, which is caused since the transmittance characteristics of
the excitation and absorption filters 16 and 21 do not match the
fluorochromes, can be reliably prevented, thereby improving the
observation performance.
Since the transmittance characteristics of the excitation and
absorption filters 16 and 21 can be continuously changed by the
single fluorescent filter set 51, an excitation switching operation
becomes unnecessary, thus improving the operability. Furthermore,
since the transmittance characteristics can be continuously
changed, the single fluorescent filter set 51 can set optimum
excitation conditions for a large number of specimens, resulting in
a reduction in the number of components and the manufacturing
costs.
various modifications of the present invention having arrangements
associated with the fluorescent filter set will be described with
reference to FIGS. 12 to 16.
In the first modification shown in FIG. 12, four different types of
fluorescent filter sets 71 to 74 are held by a turret having a
rotating axis .circle. parallel to the observation optical axis, so
that an arbitrary fluorescent filter set can be removed from and
inserted in the observation optical path. FIG. 13 shows the section
of the fluorescent filter set 71 disposed on the observation
optical path.
The respective fluorescent filter sets 71 to 74 have different
transmittance characteristics, and the arrangement of each
fluorescent filter set is substantially the same as that of the
fluorescent filter set 51 described in the third embodiment. More
specifically, an excitation filter 16 is disposed in the light
source-side opening of a fluorescent cube 60 together with a
rotating shaft 62 to be pivotal about the rotating shaft 62, and an
absorption filter 21 is disposed in the prism-side opening of the
through port of the fluorescent cube 60 together with a rotating
shaft 64 to be pivotal about the rotating shaft 64. A gear 63 is
concentrically fixed to one end of the rotating shaft 62, and a
gear 65 is concentrically fixed to one end of the rotating shaft
64. The rotating shafts 62 and 64 project from a side surface of
the fluorescent cube 60. The gears 63 and 65 are coupled by a gear
66.
According to this modification having the arrangement as described
above, the transmittance characteristics of excitation and
absorption filters 16 and 21 can be continuously changed by the
fluorescent filter sets 71 to 74. In addition, when the wavelengths
of light emitted by fluorochromes are largely different, the turret
is rotated to dispose a fluorescent filter set having appropriate
transmittance characteristics on the optical path, thereby
obtaining optimum excitation conditions.
In the second modifications shown in FIGS. 14 and 15, three
different types of fluorescent filter sets 81 to 83 can be
removably inserted in the observation optical path, and excitation
and absorption filters of each fluorescent filter set are
interlocked by a belt.
In this modification, a guide member 84 is fixed at a location away
from a location where each fluorescent filter set is to be disposed
by a predetermined distance. A guide groove is formed in a surface
of the guide member 84 opposing the observation optical axis to
extend in a direction perpendicular to the observation optical
axis. A slider 85 is mounted on the guide member 84 by slidably
fitting a linear guide portion 85a in the guide groove. The three
types of fluorescent filter sets 81 to 83 are mounted and fixed on
the other surface of the slider 85 equidistantly.
Although the transmittance characteristics of the fluorescent
filter sets 81 to 83 are different, their structures are the same.
The structure of the fluorescent filter set 81 will be described as
an example.
A fluorescent cube 86 has a shape similar to that of the tube body
31 described in the first embodiment. An excitation filter 16,
integral with a rotating shaft 87, is supported in the light
source-side opening of the fluorescent cube 86 to be pivotal about
the rotating shaft 87. A pulley 88 is mounted on the rotating shaft
87.
An absorption filter 21, integral with a rotating shaft 89, is
supported in the prism-side opening of the through port of the
fluorescent cube 86 to be pivotal about the rotating shaft 89. A
pulley 90 is mounted on the rotating shaft 89. A belt 91 extends
between the two pulleys 88 and 90.
The ratio of the diameters of the pulleys 88 and 90 is set such
that the shift amounts of the cutoff wavelengths obtained by
inclining the excitation and absorption filters 16 and 21 with
respect to the corresponding optical axes become equal to each
other.
In the modification having the arrangement as described above, a
desired fluorescent filter set (81, 82, or 83) is disposed on the
observation optical path by moving the slider 85 along the guide
member 84 in a direction perpendicular to the observation optical
axis.
When the transmittance characteristics of the excitation and
absorption filters 16 and 21 must be adjusted, the belt 91 is
driven by a driving mechanism (not shown). Then, the excitation and
absorption filters 16 and 21 are pivoted in the interlocked manner,
so that the corresponding transmittance characteristics are shifted
in predetermined directions, thereby setting optimum
conditions.
In the third modification shown in FIG. 16, excitation and
absorption filters 16 and 21 are inclined in the interlocked manner
by electrically driving them.
In this modification, stepper motors 92a and 92b for independently
driving the excitation and absorption filters 16 and 21,
respectively, of a fluorescent filter set are provided to a
fluorescent cube 90.
Rotary angles of the excitation and absorption filters 16 and 21
with which the shift amounts of the cutoff wavelengths of the
excitation and absorption filters 16 and 21 become equal to each
other are stored in a memory 93. When an excitation wavelength
shift signal is input from an encoder 94 to a CPU 95, an interlock
circuit 96 calculates the driving amounts of the excitation and
absorption filters 16 and 21 with which the shift amounts of the
cutoff wavelengths of the excitation and absorption filters 16 and
21 become equal to each other.
When the calculation results obtained by the interlock circuit 96
are output to drivers 97 and 98 to drive the stepper motors 92a and
92b, respectively, the excitation and absorption filters 16 and 21
can be inclined by desired amounts in the interlocked manner.
In this modification, since the rotary angle data are stored in the
memory 93, even if the interlocked relation between the excitation
and absorption filters 16 and 21 is not constant, an arbitrary
control operation can be performed in accordance with the rotary
angle data.
The present invention is not limited to the embodiments and
modifications described above, and various other modifications can
be made without departing from the spirit and scope of the
invention.
* * * * *